Amino-propanol derivatives as sphingosine-1-phosphate receptor modulator

ABSTRACT

Compounds of formula (I) 
                         
wherein R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , X, Y and n are as defined in the specification, processes for their production, their uses and pharmaceutical compositions containing them.

The present invention relates to amino-propanol derivatives, process fortheir production, their uses and pharmaceutical compositions containingthem.

More particularly, the invention provides a compound of formula I

wherein

-   R₁ is C₁₋₆alkyl; C₁₋₆alkyl substituted by hydroxy, C₁₋₂alkoxy or 1    to 6 fluorine atoms; C₂₋₆alkenyl; or C₂₋₆alkynyl;    each of X, Y independently is O, CH₂, or C═O;-   R₂ is phenyl; naphthyl; C₃₋₆cycloalkyl; heteroaryl; a heterocyclic    residue; phenylC₁₋₂alkyl; C₃₋₆cycloalkylC₁₋₂alkyl;    heteroarylC₁₋₂alkyl; heterocyclicC₁₋₂alkyl residue; wherein each may    be ring-substituted by 1 to 5 substituents selected from hydroxy,    halogen, C₁₋₄alkyl, C₁₋₄alkyl substituted by 1 to 5 fluorine atoms,    C₃₋₆cycloalkyl, C₁₋₄alkoxy, C₁₋₄alkoxy substituted by 1 to 5    fluorine atoms, C₃₋₆cycloalkoxy, C₃₋₆cycloalkylC₁₋₂alkyl, cyano,    phenyl, and phenyl substituted by hydroxy, halogen, C₁₋₄alkyl,    C₁₋₄alkyl substituted by 1 to 5 fluorine atoms, C₁₋₄alkoxy,    C₁₋₄alkoxy substituted by 1 to 5 fluorine atoms, or cyano;-   R₃ is Z—X₂ wherein Z is CH₂, CHF or CF₂ and X₂ is OH or a residue of    formula (a)

-   -   wherein Z₁ is a direct bond, CH₂, CHF, CF₂ or O, and each of R₈        and R₉, independently, is H or C₁₋₄alkyl optionally substituted        by 1, 2 or 3 halogen atoms; and

-   each of R₄ and R₅, independently, is H; C₁₋₄alkyl optionally    substituted by 1, 2 or 3 halogen atoms; or acyl;

-   each of R₆ and R₇, independently, is H; hydroxy; halogen; C₁₋₄alkyl;    C₁₋₆cycloalkyl; C₁₋₄alkoxy; C₁₋₆cycloalkoxy;    C₃₋₆cycloalkylC₁₋₂alkyl; or cyano; and

-   n is 2 or 3;

-   in free form or in salt form.

Alkyl or alkyl moiety may be straight or branched chain. Alkenyl may bee.g. vinyl. Alkynyl may be e.g. propyn-2-yl. Acyl may be a residue R—COwherein R is C₁₋₆alkyl, C₃₋₆cycloalkyl, phenyl or phenyl C₁₋₄-alkyl.Halogen may be fluorine, chlorine or bromine, preferably fluorine orchlorine. When alkyl is substituted by hydroxy, it is preferably on theterminal carbon atom. PhenylC₁₋₂alkyl may be e.g. benzyl.

Heteroaryl may be a 5 to 8 membered aromatic ring comprising 1 to 4heteroatoms selected from N, O and S, e.g. pyridyl, pyrimidinyl,pyrazinyl, furyl, oxazolyl, isoxazolyl, thienyl, thiazolyl,isothiazolyl, pyrrolyl, imidazolyl, or pyrazolyl.

By heterocyclic residue is meant a 3 to 8, preferably 5 to 8, memberedsaturated or unsaturated heterocyclic ring comprising 1 to 4 heteroatomsselected from N, O and S, e.g. tetrahydrofuryl, tetrahydropyranyl,aziridinyl, piperidinyl, pyrrolidinyl, piperazinyl. The heterocyclicresidue may also be fused to an optionally substituted aryl orheteroaryl, e.g. phenyl, wherein Y is bound to said aryl or heteroaryl;examples include e.g. benzo[1,3]dioxolyl.

Compounds of formula I may exist in free form or in salt form, e.g.addition salts with e.g. inorganic acids, such as hydrochloride,hydrobromide or sulfate, salts with organic acids, such as acetate,fumarate, maleate, benzoate, citrate, malate, methanesulfonate orbenzenesulfonate salts. Compounds of formula I and their salts, inhydrate or solvate form are also part of the invention.

When the compounds of formula I have asymetric centers in the molecule,various optical isomers are obtained. The present invention alsoencompasses enantiomers, racemates, diastereoisomers and mixturesthereof. For example, the central carbon atom bearing R₁, R₃ and NR₄R₅may have the R or S configuration. Compounds having the following3-dimensional configuration are generally preferred:

Moreover, when the compounds of formula I include geometric isomers, thepresent invention embraces cis-compounds, trans-compounds and mixturesthereof. Similar considerations apply in relation to starting materialsexhibiting asymetric carbon atoms or unsaturated bonds as mentionedabove, e.g. compounds of formula II, III or IV as indicated below.

In the compounds of formula I, the following significances are preferredindividually or in any sub-combination:

-   1. X is O;-   2. Y is O or S;-   3. R₁ is CH₃ or CH₂—OH;-   4. R₂ is phenyl; phenyl substituted by 1 or 2 substituents selected    from halogen, C₁₋₂alkyl or C₁₋₂alkoxy; cyanophenyl; naphthyl; or    benzo[1,3]dioxol-5-yl;-   5. R₃ is CH₂—OH or CH₂—OPO₃H₂;-   6. each of R₄ and R₅ is hydrogen;-   7. R₆ is hydrogen, methoxy, methyl, or chloro; and-   8. R₇ is hydrogen, methoxy, methyl, or chloro.

The present invention also includes a process for the preparation of acompound of formula I which process comprises

a) for a compound of formula I wherein R₃ is Z—X₂, X₂ being OH, removingthe protecting group present in a compound of formula II

wherein X, n, R₁, R₂ and R₄ are as defined above, R′₃ is Z—X₂ wherein X₂is OH and R′₅ is an amino protecting group, orb) for a compound of formula I wherein R₃ is Z—X₂, X₂ being a residue offormula (a), removing the protecting groups present in a compound offormula III

wherein X, n, R₁, R₂, R₄ and R′₅ are as defined above, and R″₃ is Z—X₂wherein X₂ is a residue of formula (a′)

wherein Z₁ is as defined above and each of R′₈ or R′₉ is a hydrolysableor hydrogenolysable group or R′₈ and R′₉ form together a divalentbridging residue optionally fused to a ring (e.g. benzene ring),and, where required, converting the compounds of formula I obtained infree form into the desired salt form, or vice versa.

Process step a) may be carried out in accordance with methods known inthe art. The removal of the amino protecting groups may conveniently beperformed according to methods known in the art, e.g. by hydrolysis,e.g. in an acidic medium, for example using hydrochloric acid. Examplesof protecting groups for amino groups are e.g. as disclosed in“Protective Groups in Organic Synthesis” T. W. Greene, J. Wiley & SonsNY, 2^(nd) ed., chapter 7, 1991, and references therein, e.g. benzyl,p-methoxybenzyl, methoxymethyl, tetrahydropyranyl, trialkylsilyl, acyl,tert.-butoxy-carbonyl, benzyloxycarbonyl, 9-fluorenyl methoxy carbonyl,trifluoroacetyl, and the like.

In the residue of formula (a′), each of R′₈ and R′₉ may have thesignificance of e.g. tert-butyl, phenyl or benzyl or form together acyclic system such as in 1,5-dihydro-2,4,3-benzodioxaphosphepin.

Process step b) may be performed according to methods known in the art,e.g. by hydrolysis, e.g. in a basic medium when R′₆ and R′₇ are each ahydrolysable group, for example using a hydroxide such as bariumhydroxide or in an acidic medium when R′₆ and R′₇ are each a tert-butylgroup. It may also be performed by hydrogenolysis, e.g. in the presenceof a catalyst, e.g. Pd/C, followed by hydrolysis, e.g. in an acidicmedium, for example HCl. Compounds of formulae II and III, used asstarting materials, and salts thereof are also novel and form part ofthe invention.

The present invention also includes a process for the preparation of acompound of formula II which process comprises transforming a compoundof formula IV

wherein R₁, R′₃, R₄ and R′₅ are as defined above, to introduce thedesired residue —(CH₂)_(n)—Y—R₂ e.g. by an alkylation. Alkylation of thecompounds of formula IV may be performed according to methods known inthe art, e.g. by nucleophilic substitution, e.g. by reaction with analkylating agent R₂—Y—(CH₂)_(n)—X₃ wherein X₃ is mesylate, tosylate,triflate, nosylate, chloride, bromide or iodide. The alkylation may alsobe carried out by following the Mitsunobu protocol usingR₂—Y—(CH₂)_(n)—OH (e.g. as disclosed in Hughes, Organic Preparations andProcedures International 28, 127-64, 1996 or D. L. Hughes, Org. React.42, 335, 1992), in solution or on solid phase support synthesis, e.g. byattaching the compound of formula IV to a resin. Alternatively, e.g.triphenylphosphine or diethyl azocarboxylate bound to a resin, e.g.polystyrene, may be used.

Compounds of formula III wherein R′₈ and R′₉ form a cyclic system, maybe prepared as follows:

wherein X, Y, n, R₁, R₂, R₄ and R′₅ are as defined above.

Insofar as the production of the starting materials is not particularlydescribed, the compounds are known or may be prepared analogously tomethods known in the art or as disclosed in the Examples hereinafter.

The following Examples are illustrative of the invention. Melting pointsare uncorrected.

RT = room temperature THF = tetrahydrofuran DMF = dimethylformamide MTBE= methyl tert.-butyl ether AcOEt = ethyl acetate

EXAMPLE 1(R)-2-Amino-4-{4-[2-(4-chloro-phenoxy)-ethoxy]-phenyl}-2-methyl-butan-1-olHydrochloride

To tert.-butyl(R-(3-{4-[2-(4-Chloro-phenoxy)-ethoxy]-phenyl}-1-hydroxymethyl-1-methyl-propylycarbamate(38 mg, 0.063 mmol) is added 4 M HCl in dry dioxane (1 mL). The clearcolorless solution is stirred for 2 h protected from moisture. Then, thesolution is evaporated to dryness and the partially crystalline residueis taken up in dry ether (5 mL). Sonication for 10 min gives aprecipitate of colorless crystals. The product is filtered off, washedwith cold ether (3×1 mL), and dried in vacuo to afford the titlecompound in form of a non hygroscopic colorless microcrystalline powder:HPLC: t_(R)=3.17 min., ESI+ MS: m/z=350/352 (MH⁺).

tert.-butyl(R)-(3-{4-[2-(4-Chloro-phenoxy)-ethoxy]-phenyl}-1-hydroxymethyl-1-methyl-propyl)-carbamatemay be prepared according to the following procedure:

To a solution of tert-butyl[(R)-1-hydroxy-4-(4-hydroxy-phenyl)-2-methyl-but-2-yl]-carbamate (100mg, 0.34 mmol) and 1-(2-bromoethoxy)-4-chlorobenzene (120 mg, 0.51 mmol,1.5 eq.) in anhydrous DMF (2.5 mL) is added water free caesium carbonate(166 mg, 0.51 mmol, 1.5 eq.). The suspension obtained is stirred overnight protected from moisture at 60° C. After cooling to RT the solidsare filtered off and rinsed with DMF (2×1 mL). The combined filtratesare evaporated in a high vacuum to give a dark orange syrup.Purification by flash chromatography (FlashMaster II, MTBE/hexanesgradient 0% MTBE→40% MTBE within 45 min, then 40% MTBE 15 min) givescolorless crystals: HPLC: t_(R)=5.45 min., ESI+ MS: m/z=450/452.

By following the procedure of Example 1 but using the appropriatestarting materials, the compounds of formula A, wherein R₂ and R₆ are asindicated in Table 1, may be prepared.

TABLE 1 A

Example R₂ R₆ M.S. Data 2. phenyl H MH⁺ 316.5 3.

H MH⁺ 376.5 4.

H MH⁺ 394.3 5.

H MH⁺ 330.4 6.

H MH⁺ 334.4 7.

H MH⁺ 352.4 8.

H MH⁺ 394.3 9.

methoxy MH⁺ 380.9 10. phenyl methoxy MH⁺ 346.5

EXAMPLE 11 2-Amino-4-{4-[2-phenoxy-ethoxy]-phenyl}-2-ethoxy-butan-1-olHydrochloride

To a solution of4-[2-(4-hydroxymethyl-2-methyl-4,5-dihydro-oxazol-4-yl)-ethyl]-phenol(300 mg, 1.27 mmol) in DMF (5 ml) was added Cs₂CO₃ (1.2 g, 3.83 mmol)and 2-phenoxyethyl bromide (770 mg, 1.27 mmol). The reaction mixture wasstirred at 85° C. for 4 hours. AcOEt and water were then added, theorganic layer was separated and the aqueous phase was extracted withAcOEt (3×50 ml). The combined organic extracts were washed with brine,dried over MgSO₄, and evaporated to dryness. Purification by flashchromatography (AcOEt/Hx 9:1) afforded(2-Methyl-4-{2-[4-(2-phenoxy-ethoxy)phenyl]-ethyl}-4,5-dihydro-oxazol-4-yl)-methanolas colorless oil. To a solution of(2-methyl-4-{2-[4-(2-phenoxy-ethoxy)phenyl]-ethyl}-4,5-dihydro-oxazol-4-yl)-methanol(50 mg, 0.14 mmol) in ethanol (2 ml) was added conc. HCl (2 ml). Thereaction mixture was stirred at 85° C. for 2 hours, then concentrated todryness. The residue was re-dissolved in AcOEt and precipitated withhexanes. The solid was filtered off, washed with dry ether and dried invacuo to afford2-amino-4-{4-[2-phenoxy-ethoxy]-phenyl}-2-ethoxy-butan-1-olhydrochloride as a colorless powder. ESI+MS: m/z=332.3 (M+H)⁺.

By following the procedure of Example 11 but using the appropriatestarting materials, the compounds of formula B, wherein R₂ is asindicated in Table 2, may be prepared.

TABLE 2 B

Example R₂ M.S. Data 12

(M + H)⁺ 410.1 13

(M + H)⁺ 410.1 14

(M + H)⁺ 382.2 15

(M + H)⁺ 362.2 16

(M + H)⁺ 376.0 17

(M + H)⁺ 366.2 18

(M + H)⁺ 346.2 19

(M + H)⁺ 362.2 20

(M + H)⁺ 362.0 21

(M + H)⁺ 400.1 22

(M + H)⁺ 376.0 23

(M + H)⁺ 357.4 24

MH⁺ 346.4 25

MH⁺ 350.4

EXAMPLE 26 (+/−)Mono-(2-Amino-4-{4-[2-phenoxy-ethoxy]-phenyl}-2-ethoxy-butan)

To a solution of(2-methyl-4-{2-[4-(2-phenoxy-ethoxy)-phenyl]-ethyl}-4,5-dihydro-oxazol-4-yl)-methanol(200 mg, 0.56 mmol) and tetrazole (197 mg, 2.81 mmol, 5 eq.,recrystallized from toluene) in dry THF (5 ml) was addeddi-tert-butyl-N,N-diisopropylphosphoramide (561 mg, 2.25 mmol, 4 eq.).After stirring under argon at RT for 3 h, H₂O₂ (30%, 10 eq.) was slowlyadded at 0° C. with vigorous stirring. The reaction mixture was stirredfor further 30 min, followed by addition of saturated sodium thiosulfatesolution (5 ml). The organic layer was separated and the aqueous phasewas extracted with ether (3×20 ml). The combined organic extracts werewashed with brine, dried over MgSO₄, and evaporated to dryness.Purification by flash chromatography (AcOEt/hexane 1:1) affordedphosphoric acid di-tert-butyl ester2-methyl-4-{2-[4-(2-phenoxy-ethoxy)-phenyl]-ethyl}-4,5-dihydro-oxazol-4-ylmethylester as colorles crystals. Finally, to a solution of phosphoric aciddi-tert-butyl ester2-methyl-4-{2-[4-(2-phenoxy-ethoxy)-phenyl]-ethyl}-4,5-dihydro-oxazol-4-ylmethylester (150 mg, 0.27 mmol) in ethanol (5 ml) was added conc. HCl (5 ml).The reaction mixture was stirred at 50° C. for 2 hours, thenconcentrated to dryness. The residue was re-dissolved in AcOEt andprecipitated with hexanes. The solid was filtered off, washed with dryether and dried in vacuo to affordmono-(2-amino-4-{4-[2-phenoxy-ethoxy]-phenyl}-2-ethoxy-butan) phosphateas a colorless powder. ESI+ MS: m/z=410.2 (M−H)⁺.

By following the procedure of Example 26 but using the appropriatestarting materials, the compounds of formula C, wherein R₂ is asindicated in Table 3, may be prepared.

TABLE 3 C

Example R₂ M.S. Data 27

(M + H)⁺ 488.0, (M − H)⁺ 490.0 28

(M + H)⁺ 488.0, (M − H)⁺ 490.0 29

(M + H)⁺ 440.2 30

(M + H)⁺ 440.2 31

(M + H)⁺ 444.0 32

(M + H)⁺ 424.0 33

MH⁺ 426.4 34

MH⁺ 430.3 35

(M + H)⁺ 442.4

EXAMPLE 36(R)-Mono-(2-amino-4-{4-[2-(4-chloro-phenoxy)-ethoxy]-phenyl}-2-methyl-butyl)phosphate

tert.-butyl[3-{4-[2-(4-Chloro-phenoxy)-ethoxy]-phenyl}-1-(di-tert-butoxy-phosphoryloxy-methyl)-1-methyl-propyl]-carbamate(44 mg, 0.069 mmol) was dissolved in 4 M HCl in dioxane (2 mL). Afterstirring for 2 h the slightly turbid solution was evaporated. Thecolorless semi-solid residue was sonicated with dry ether (5 mL) to givea colorless precipitate. The solid was filtered off, washed with dryether and vacuum dried to afford a off white powder: HPLC: t_(R)=3.11min., ESI+ MS: m/z=430/432 (M−H⁺).

tert.-butyl[3-{4-[2-(4-Chloro-phenoxy)-ethoxy]-phenyl}-1-(di-tert-butoxy-phosphoryloxy-methyl)-1-methyl-propyl]-carbamatebe prepared according to the following procedure:

To a solution of tert.-butyl(R)-(3-{4-[2-(4-Chloro-phenoxy)-ethoxy]-phenyl}-1-hydroxymethyl-1-methyl-propyl)-carbamate(40 mg, 0.089 mmol, Ex. 1a) and tetrazole (19 mg, 0.27 mmol, 3 eq.,recrystallized from toluene) in dry THF (1 mL) was added di-tert.-butylN,N-diethyl-phosphoramidite (36 μL, 0.13 mmol, 1.5 eq.). The reactionmixture was stirred under argon at RT for 2 h. Then, H₂O₂ (30%, 91 μL,0.89 mmol, 10 eq.) was injected at 0° C. with vigorous stirring. Thereaction mixture was stirred for further 30 min, followed by addition ofsaturated sodium thiosulfate solution (1 mL). The organic layer wasseparated and the aqueous phase was extracted with ether (3×1 mL). Thecombined organic extracts were washed with brine, dried over MgSO₄, andevaporated to dryness. The crude material was purified by flashchromatography (FlashMaster II, MTBE/hexanes gradient 0% MTBE→50% MTBEwithin 45 min, then 50% MTBE 15 min) to afford colorless crystals: HPLC:t_(R)=6.64 min., ESI+ MS: m/z=642/644 (MH⁺).

By following the procedure of Example 36 but using the appropriatestarting materials, the compounds of formula D, wherein R₂ and R₆ are asindicated in Table 4, may be prepared.

TABLE 4 D

Example R₂ R₆ M.S. Data 38 phenyl H (M + H)⁺ 396.3 39

H MH⁺ 472.0 40

H MH⁺ 408.2 41

H MH⁺ 412.1 42

H MH⁺ 432.1 43 phenyl methoxy M − H 424.4

EXAMPLE 442-Amino-2-{2-[4-(3-phenoxy-propoxy)-phenyl]-ethyl}-propane-1,3-diolHydrochloride

To a solution of4-[2-(4-hydroxymethyl-2-methyl-4,5-dihydro-oxazol-4-yl)-ethyl]-phenol(300 mg, 1.27 mmol) in DMF (5 ml) was added Cs₂CO₃ (1.2 g, 3.83 mmol)and (3-bromo-propoxy)-benzene (273 mg, 1.27 mmol). The reaction mixturewas stirred at 85° C. for 4 hours. AcOEt and water were then added, theorganic layer was separated and the aqueous phase was extracted withAcOEt (3×50 ml). The combined organic extracts were washed with brine,dried over MgSO₄, and evaporated to dryness. Purification by flashchromatography (AcOEt/Hexane 9:1) affords(2-methyl-4-{2-[4-(3-phenoxy-propoxy)-phenyl]-ethyl}-4,5-dihydro-oxazol-4-yl)-methanolas colorless oil. To a solution of(2-methyl-4-{2-[4-(3-phenoxy-propoxy)-phenyl]-ethyl}-4,5-dihydro-oxazol-4-yl)-methanol(50 mg, 0.135 mmol) in ethanol (2 ml) was added conc. HCl (2 ml). Thereaction mixture was stirred at 85° C. for 2 hours, then concentrated todryness. The residue was re-dissolved in AcOEt and precipitated withhexanes. The solid was filtered off, washed with dry ether and dried invacuo to afford2-amino-2-{2-[4-(3-phenoxy-propoxy)-phenyl]-ethyl}-propane-1,3-diolhydro-chloride as a colorless powder. ESI+ MS: m/z=370.4 (M+H)⁺.

By following the procedure of Example 44 but using the appropriatestarting materials, the compounds of formula E, wherein R₂ and Y are asindicated in Table 5, may be prepared.

TABLE 5 E

Example R₂ Y M.S. Data 45 3-MeO-phenyl O (M + H)⁺ 376.4 46 2-Br-phenyl O(M + H)⁺ 425.3 47 4-Cl-phenyl O (M + H)⁺ 380.9 48

O (M + H)⁺ 455.3 49 3-Me-phenyl O (M + H)⁺ 360.4 50 4-MeO-phenyl O (M +H)⁺ 376.4 51 phenyl S (M + H)⁺ 362.5

EXAMPLE 52((R)-2-Amino-2-methyl-4-[4-(3-phenoxy-propoxy)phenyl]-butan-1-olHydrochloride

To{(R)-1-Hydroxymethyl-1-methyl-3-[4-(3-phenoxy-propoxy)-phenyl]-propyl}-carbamicacid tert-butyl (50 mg, 0.11 mmol) was added 4 M HCl in dry dioxane (1mL). The clear colorless solution was stirred for 2 h protected frommoisture. Then, the solution was evaporated to dryness and the partiallycrystalline residue was taken up in dry ether (5 mL). Sonication for 10min gives a precipitate of colorless crystals. The product was filteredoff, washed with cold ether (3×1 mL), and dried in vacuo to afford thetitle compound in form of a non hygroscopic colorless microcrystallinepowder. ESI+MS: m/z=330.4 (MH⁺).

{(R)-1-Hydroxymethyl-1-methyl-3-[4-(3-phenoxy-propoxy)-phenyl]-propyl}-carbamicacid tert-butyl may be prepared according to the following procedure:

To a solution of tert-butyl[(R)-1-hydroxy-4-(4-hydroxy-phenyl)-2-methyl-but-2-yl]-carbamate (100mg, 0.34 mmol) and (3-bromo-propoxy)-benzene (109.6 mg, 0.51 mmol, 1.5eq.) in anhydrous DMF (2.5 mL) was added water free cesium carbonate(166 mg, 0.51 mmol, 1.5 eq.). The suspension obtained was stirred overnight protected from moisture at 60° C. After cooling to RT the solidswere filtered off and rinsed with DMF (2×1 mL). The combined filtrateswere evaporated in a high vacuum to give a dark orange syrup.Purification by flash chromatography (FlashMaster II, MTBE/hexanesgradient 0% MTBE→40% MTBE within 45 min, then 40% MTBE 15 min) gavecolorless crystals: ESI+MS: m/z=430.4.

EXAMPLE 53(R)-2-Amino-4-{4-[3-(5-bromo-2-methoxy-phenoxy)-propoxy]-phenyl}-2-methyl-butan-1-olHydrochloride

The title compound was prepared as described in example 52 using4-bromo-2-(3-bromo propoxy)-1-methoxy-benzene instead of(3-bromo-propoxy)-benzene. ESI+ MS: m/z=439.3 (M+H)⁺.

EXAMPLE 54 (+/−) Mono Phosphoric acidmono-{2-amino-2-hydroxymethyl-4-[4-(3-phenoxy-propoxy)-phenyl]-butyl}ester

To a solution of(2-methyl-4-{2-[4-(3-phenoxy-propoxy)-phenyl]-ethyl}-4,5-dihydro-oxazol-4-yl)-methanol(200 mg, 0.54 mmol) and tetrazole (189 mg, 2.70 mmol, 5 eq.,recrystallized from toluene) in dry THF (5 ml) was addeddi-tert-butyl-N,N-diisopropylphosphoramide (538.5 mg, 2.16 mmol, 4 eq.).After stirring under argon at RT for 3 h, H₂O₂ (30%, 10 eq.) was slowlyadded at 0° C. with vigorous stirring. The reaction mixture was stirredfor further 30 min, followed by addition of saturated sodium thiosulfatesolution (5 ml). The organic layer was separated and the aqueous phasewas extracted with ether (3×20 ml). The combined organic extracts werewashed with brine, dried over MgSO₄, and evaporated to dryness.Purification by flash chromatography (AcOEt/hexane 1:1) affords (+/−)phosphoric acid di-tert-butyl ester2-methyl-4-{2-[4-(3-phenoxy-propoxy)-phenyl]-ethyl}-4,5-dihydro-oxazol-4-ylmethylester as colorless crystals. Finally, to a solution of phosphoric aciddi-tert-butyl ester2-methyl-4-{2-[4-(3-phenoxy-propoxy)-phenyl]-ethyl}-4,5-dihydro-oxazol-4-yl-methylester (50 mg, 0.089 mmol) in ethanol (5 ml) was added conc. HCl (5 ml).The reaction mixture was stirred at 50° C. for 2 hours, thenconcentrated to dryness. The residue was re-dissolved in AcOEt andprecipitated with hexanes. The solid was filtered off, washed with dryether and dried in vacuo to afford (+/−) mono phosphoric acidmono-{2-amino-2-hydroxymethyl-4-[4-(3-phenoxy-propoxy)phenyl]-butyl}ester as a colorless powder. ESI+MS: m/z=426.4 (M−H)⁺.

By following the procedure of Example 54 but using the appropriatestarting materials, the compounds of formula F, wherein R₂, R₁₀ and Yare as indicated in Table 6, may be prepared.

TABLE 6 F

Example R₂ R₁₀ Y M.S. Data 55 2-Br-phenyl CH₂—OH O (M + H)⁺ 505.3 56

CH₂—OH O (M + H)⁺ 535.3 57 phenyl CH₂—OH S (M + H)⁺ 442.4 58

CH₃ O (M + H)⁺ 519.3

The compounds of formula I in free form or in pharmaceuticallyacceptable salt form, exhibit valuable pharmacological properties, e.g.lymphocyte recirculation modulating properties, e.g. as indicated in invitro and in vivo tests and are therefore indicated for therapy.

A. In Vitro

The compounds of formula I have binding affinity to individual human SIPreceptors as determined in following assays:

Sphingosine-1-Phosphate (S1P) Receptor Profiling

Agonist activities of compounds are tested on the human SIP receptorsEDG-1 (S1P₁), EDG-3 (S1P₃), EDG-5 (S1P₂), EDG-6 (S1P₄) and EDG-8 (S1P₅).Functional receptor activation is assessed by quantifying compoundinduced GTP [γ-³⁵S] binding to membrane protein prepared fromtransfected CHO or RH7777 cells stably expressing the appropriate humanS1P receptor. The assay technology used is SPA (scintillation proximitybased assay). Briefly, DMSO dissolved compounds are serially diluted andadded to SPA-bead (Amersham-Pharmacia) immobilised SIP receptorexpressing membrane protein (10-20 μg/well) in the presence of 50 mMHepes, 100 mM NaCl, 10 mM MgCl₂, 10 μM GDP, 0.1% fat free BSA and 0.2 nMGTP [γ-³⁵S] (1200 Ci/mmol). After incubation in 96 well microtiterplatesat RT for 120 min, unbound GTP [γ-³⁵S] is separated by a centrifugationstep. Luminescence of SPA beads triggered by membrane bound GTP [γ-³⁵S]is quantified with a TOP count plate reader (Packard). EC₅₀s arecalculated using standard curve fitting software. In this assay, thecompounds of formula I have a binding affinity to S1P₁ receptor <50 nM.

S1P₁ S1P₃ S1P₄ S1P₅ Example EC₅₀ [nM] EC₅₀ [nM] EC₅₀ [nM] EC₅₀ [nM] 360.6 Agon. 15 Agon. >1000 14 Agon. 38 8.6 Agon. >1000 28 Agon. 11.7 Agon.42 5.5 Agon. >1000 8.2 Agon. 6.3 Agon. 43 8 Agon. 340 Agon. 43 Agon. 0.5Agon. 56 8.5 Agon. >10000 Agon. 1.6 Agon. 16.5 Agon Agon. = agonistB. In Vivo: Blood Lymphocyte Depletion

A compound of formula I or the vehicle is administered orally by gavageto rats. Tail blood for hematological monitoring is obtained on day −1to give the baseline individual values, and at 2, 6, 24, 48 and 72 hoursafter application. In this assay, the compounds of formula I depleteperipheral blood lymphocytes when administered at a dose of 0.03 to 3mg/kg. For example, following results are obtained: depletion ofperipheral blood lymphocytes by more than 50%

Example 1: 0.03 mg/kg p.o. after 6 h, 0.02 mg/kg p.o. after 24 h.Example 3: 0.01 mg/kg p.o. after 6 h, <0.03 mg/kg p.o. after 48 h.Example 7: 0.03 mg/kg p.o. after 6 h, 0.02 mg/kg p.o. after 48 h.Example 9: 0.2 mg/kg p.o. after 6 h, >1 mg/kg p.o. after 48 h. Example53: 0.1 mg/kg p.o. after 6 h, 0.5 mg/kg p.o. after 48 h.

The compounds of formula I are, therefore, useful in the treatmentand/or prevention of diseases or disorders mediated by lymphocytesinteractions, e.g. in transplantation, such as acute or chronicrejection of cell, tissue or organ allo- or xenografts or delayed graftfunction, graft versus host disease, autoimmune diseases, e.g.rheumatoid arthritis, systemic lupus erythematosus, hashimoto'sthyroidis, multiple sclerosis, myasthenia gravis, diabetes type I or IIand the disorders associated therewith, vasculitis, pernicious anemia,Sjoegren syndrome, uveits, psoriasis, Graves ophthalmopathy, alopeciaareata and others, allergic diseases, e.g. allergic asthma, atopicdermatitis, allergic rhinitis/conjunctivitis, allergic contactdermatitis, inflammatory diseases optionally with underlying aberrantreactions, e.g. inflammatory bowel disease, Crohn's disease orulcerative colitis, intrinsic asthma, inflammatory lung injury,inflammatory liver injury, inflammatory glomerular injury,atherosclerosis, osteoarthritis, irritant contact dermatitis and furthereczematous dermatitises, seborrhoeic dermatitis, cutaneousmanifestations of immunologically-mediated disorders, inflammatory eyedisease, keratoconjunctivitis, myocarditis or hepatitis,ischemia/reperfusion injury, e.g. myocardial infarction, stroke, gutischemia, renal failure or hemorrhage shock, traumatic shock,angiogenesis, Alzheimer's disease, cancer, e.g. breast cancer, T celllymphomas or T cell leukemias, infectious diseases, e.g. toxic shock(e.g. superantigen induced), septic shock, adult respiratory distresssyndrome or viral infections, e.g. AIDS, viral hepatitis, chronicbacterial infection, or senile dementia. Examples of cell, tissue orsolid organ transplants include e.g. pancreatic islets, stem cells, bonemarrow, corneal tissue, neuronal tissue, heart, lung, combinedheart-lung, kidney, liver, bowel, pancreas, trachea or oesophagus. Forthe above uses the required dosage will of course vary depending on themode of administration, the particular condition to be treated and theeffect desired.

In general, satisfactory results are indicated to be obtainedsystemically at daily dosages of from about 0.03 to 2.5 mg/kg per bodyweight. An indicated daily dosage in the larger mammal, e.g. humans, isin the range from about 0.5 mg to about 100 mg, convenientlyadministered, for example, in divided doses up to four times a day or inretard form. Suitable unit dosage forms for oral administration comprisefrom ca. 0.1 to 50 mg active ingredient.

The compounds of formula I may be administered by any conventionalroute, in particular enterally, e.g. orally, e.g. in the form of tabletsor capsules, or parenterally, e.g. in the form of injectable solutionsor suspensions, topically, e.g. in the form of lotions, gels, ointmentsor creams, or in a nasal or a suppository form. Pharmaceuticalcompositions comprising a compound of formula I in free form or inpharmaceutically acceptable salt form in association with at least onepharmaceutical acceptable carrier or diluent may be manufactured inconventional manner by mixing with a pharmaceutically acceptable carrieror diluent.

The compounds of formula I may be administered in free form or inpharmaceutically acceptable salt form e.g. as indicated above. Suchsalts may be prepared in conventional manner and exhibit the same orderof activity as the free compounds.

In accordance with the foregoing the present invention further provides:

-   1.1 A method for preventing or treating disorders or diseases    mediated by lymphocytes, e.g. such as indicated above, in a subject    in need of such treatment, which method comprises administering to    said subject an effective amount of a compound of formula I or a    pharmaceutically acceptable salt thereof;-   1.2 A method for preventing or treating acute or chronic transplant    rejection or T-cell mediated inflammatory or autoimmune diseases,    e.g. as indicated above, in a subject in need of such treatment,    which method comprises administering to said subject an effective    amount of a compound of formula I or a pharmaceutically acceptable    salt thereof;-   2. A compound of formula I, in free form or in a pharmaceutically    acceptable salt form for use as a pharmaceutical, e.g. in any of the    methods as indicated under 1.1 or 1.2 above.-   3. A pharmaceutical composition, e.g. for use in any of the methods    as in 1.1 or 1.2 above comprising a compound of formula I in free    form or pharmaceutically acceptable salt form in association with a    pharmaceutically acceptable diluent or carrier therefor.-   4. A compound of formula I or a pharmaceutically acceptable salt    thereof for use in the preparation of a pharmaceutical composition    for use in any of the method as in 1.1 or 1.2 above.

The compounds of formula I may be administered as the sole activeingredient or in conjunction with, e.g. as an adjuvant to, other drugse.g. immunosuppressive or immunomodulating agents or otheranti-inflammatory agents, e.g. for the treatment or prevention of allo-or xenograft acute or chronic rejection or inflammatory or autoimmunedisorders, or a chemotherapeutic agent, e.g a malignant cellanti-proliferative agent. For example, the compounds of formula I may beused in combination with a calcineurin inhibitor, e.g. cyclosporin A, FK506 or ISA_(TX)247; a mTOR inhibitor, e.g. rapamycin,40-O-(2-hydroxyethyl)-rapamycin, CCI779, ABT578, AP23573, AP23464,AP23675, AP23841, TAFA-93, biolimus 7 or biolimus 9; an ascomycin havingimmunosuppressive properties, e.g. ABT-281, ASM981, etc.;corticosteroids; cyclophosphamide; azathioprene; methotrexate;leflunomide; mizoribine; mycophenolic acid; mycophenolate mofetil;15-deoxyspergualine or an immunosuppressive homologue, analogue orderivative thereof; immunosuppressive monoclonal antibodies, e.g.,monoclonal antibodies to leukocyte receptors, e.g., MHC, CD2, CD3, CD4,CD7, CD8, CD25, CD28, CD40. CD45, CD58, CD80, CD86 or their ligands;other immunomodulatory compounds, e.g. a recombinant binding moleculehaving at least a portion of the extracellular domain of CTLA4 or amutant thereof, e.g. an at least extracellular portion of CTLA4 or amutant thereof joined to a non-CTLA4 protein sequence, e.g. CTLA4Ig (forex. designated ATCC 68629) or a mutant thereof, e.g. LEA29Y; adhesionmolecule inhibitors, e.g. LFA-1 antagonists, ICAM-1 or -3 antagonists,VCAM-4 antagonists or VLA-4 antagonists; or a chemotherapeutic agent,e.g. paclitaxel, gemcitabine, cisplatinum, doxorubicin or5-fluorouracil; or an anti-infectious agent.

Where the compounds of formula I are administered in conjunction withother immuno-suppressive/immunomodulatory, anti-inflammatory.chemotherapeutic or anti-infectious therapy, dosages of theco-administered immunosuppressant, immunomodulatory, anti-inflammatory,chemotherapeutic or anti-infectious compound will of course varydepending on the type of co-drug employed, e.g. whether it is a steroidor a calcineurin inhibitor, on the specific drug employed, on thecondition being treated and so forth. In accordance with the foregoingthe present invention provides in a yet further aspect:

-   5. A method as defined above comprising co-administration, e.g.    concomitantly or in sequence, of a therapeutically effective    non-toxic amount of a compound of formula I and at least a second    drug substance, e.g. an immunosuppressant, immunomodulatory,    anti-inflammatory or chemotherapeutic drug, e.g. as indicated above.-   6. A pharmaceutical combination, e.g. a kit, comprising a) a first    agent which is a compound of formula I as disclosed herein, in free    form or in pharmaceutically acceptable salt form, and b) at least    one co-agent, e.g. an immunosuppressant, immunomodulatory,    anti-inflammatory, chemotherapeutic or anti-infectious agent. The    kit may comprise instructions for its administration.

The terms “co-administration” or “combined administration” or the likeas utilized herein are meant to encompass administration of the selectedtherapeutic agents to a single patient, and are intended to includetreatment regimens in which the agents are not necessarily administeredby the same route of administration or at the same time.

The term “pharmaceutical combination” as used herein means a productthat results from the mixing or combining of more than one activeingredient and includes both fixed and non-fixed combinations of theactive ingredients. The term “fixed combination” means that the activeingredients, e.g. a compound of formula I and a co-agent, are bothadministered to a patient simultaneously in the form of a single entityor dosage. The term “non-fixed combination” means that the activeingredients, e.g. a compound of formula I and a co-agent, are bothadministered to a patient as separate entities either simultaneously,concurrently or sequentially with no specific time limits, wherein suchadministration provides therapeutically effective levels of the 2compounds in the body of the patient. The latter also applies tococktail therapy, e.g. the administration of 3 or more activeingredients.

1. A compound of formula I

wherein R₁ is C₁₋₆alkyl; C₁₋₆alkyl substituted by hydroxy, C₁₋₂alkoxy or1 to 6 fluorine atoms; C₂₋₆alkenyl; or C₂₋₆alkynyl; X is O; Y is O; R₂is phenyl; naphthyl; C₃₋₆cycloalkyl; heteroaryl; a heterocyclic residue;phenyl-C₁₋₂alkyl; C₃₋₆cycloalkylC₁₋₂alkyl; heteroarylC₁₋₂alkyl;heterocyclic-C₁₋₂alkyl residue; wherein each may be ring-substituted by1 to 5 substituents selected from hydroxy, halogen, C₁₋₄alkyl, C₁₋₄alkylsubstituted by 1 to 5 fluorine atoms, C₃₋₆cycloalkyl, C₁₋₄alkoxy,C₁₋₄alkoxy substituted by 1 to 5 fluorine atoms, C₃₋₆cycloalkoxy,C₃₋₆cycloalkylC₁₋₂alkyl, cyano, phenyl, and phenyl substituted byhydroxy, halogen, C₁₋₄alkyl, C₁₋₄alkyl substituted by 1 to 5 fluorineatoms, C₁₋₄alkoxy, C₁₋₄alkoxy substituted by 1 to 5 fluorine atoms, orcyano, and where heteroaryl is a 5-to-8-membered aromatic ringcomprising 1-to-4 heteroatoms selected from N, O and S, and heterocyclicis a 3-to-8-membered saturated heterocyclic ring comprising 1-to-4heteroatoms selected from O, N and S, which ring may be fused to aphenyl; R₃ is Z—X₂ wherein Z is CH₂, CF₂ or CF₂ and X₂ is OH or aresidue of formula (a)

wherein Z₁ is a direct bond, CH₂, CHF, CF₂ or O, and each of R₈ and R₉,independently, is H or C₁₋₄alkyl optionally substituted by 1, 2 or 3halogen atoms; and each of R₄ and R₅, independently, is H; C₁₋₄alkyloptionally substituted by 1, 2 or 3 halogen atoms; or R—CO, where R isC₁₋₆-alkyl, C₃₋₆-cycloalkyl, phenyl or phenyl-C₁₋₄-alkyl; each of R₆ andR₇, independently, is H; hydroxy; halogen; C₁₋₄alkyl; C₁₋₆cycloalkyl;C₁₋₄alkoxy; C₃₋₆-cycloalkoxy; C₃₋₆cycloalkylC₁₋₂alkyl; or cyano; and nis 2 or 3; in free form or in salt form.
 2. A compound of formula Iwherein R₂ is phenyl; phenyl substituted by 1 or 2 halogen, C₁₋₂alkyl orC₁₋₂alkoxy; cyanophenyl; naphthyl; or benzo[1,3]dioxol-5-yl, in freeform or in salt form.
 3. A compound according to claim 1 wherein each ofR₆ and R₇, independently, is hydrogen, methoxy, methyl, or chloro infree form or in salt form.
 4. A compound according to claim 1 wherein R₃is CH₂—OH or CH₂—OPO₃H₂.
 5. A pharmaceutical composition comprising acompound according to claim 1, or a pharmaceutically acceptable saltthereof in association with a pharmaceutically acceptable diluent orcarrier therefore.
 6. A pharmaceutical combination comprising a compoundaccording to claim 1, in free form or in pharmaceutically acceptablesalt form, and at least one co-agent.
 7. A compound according to claim 1wherein R₁ is CH₃ or CH₂—OH.
 8. A compound according to claim 1 whereineach of R₄ and R₅ is hydrogen.